The development of effective prophylactic and therapeutic agents for controlling microorganism either planktonic or organized in biofilms adherent to cell surfaces has proven problematic.
Prophylactic and therapeutic formulations and methods developed for the prevention of infections by control of the ecological microbial balance, in general, have only been partially successful.
In nutrient-limited ecosystems, such as the aquatic environment, bacteria have a marked tendency to attach to surfaces and initiate the formation of a biofilm. The biofilm is a collection of microcolonies with water channels in between and an assortment of cells and extra cellular polymers (glycoproteins, polysaccharides and proteins). These biofilms are also a severe problem in medical science, such as in oral health where they can cause dental plaque and periodontitis.
It is well known that natural antimicrobial agents are contained in most natural external mammalian secretions. In particular, the naturally occurring antimicrobial thiocyanate/peroxidase/H2O2 systems, lactoferrin, lactoferrin peptides, lysozyme and immunoglobulins present in secretion liquids have been extensively studied.
Antimicrobial thiocyanate/peroxidase/H2O2 systems imitate the effect of the peroxidases (sialoperoxidase and myeloperoxidase) which catalyze the transformation of halide or pseudo-halide into hypohalide or pseudo hypohalite in the presence of the hydrogen peroxide produced by some bacterial strains.
Lactoferrin is bacteriostatic by fixing ferric iron and making it unavailable for bacteria metabolism. However, when the microorganisms are organized in biofilm and that the biofilm can be protected by other biofilm layers, the lactoferrin has no or not sufficient antibacterial effect against the lower layer.
Lysozyme hydrolyses proteoglycans in the bacterial cell walls causing cell lysis. When the microorganisms are organized in biofilms and are hidden by several layers of biofilms, lysozyme alone or in combination with other antimicrobial agents such as lactoferrin has no effect against these microorganisms.
Immunoglobulins are able to react specifically against the microorganisms individually. The presence of several layers of biofilms and the characteristics of the biofilms avoid the action of the immunoglobulins alone or in combination with other antimicrobial agents such as lactoferrin to react against the individual microorganisms.
The ability of the antimicrobial agents contained in saliva to react significantly on the bacteria organized in biofilms depends largely on the thickness of the biofilm. In many cases, in the presence of several layers of biofilms, the upper layer protects the lower layer against the action of the naturally occurring antimicrobial agents described above. This lower layer contains H2O2 producers and will thus be responsible for the damage of the tissue composed of epithelial and fibroblast cells.
Some of these antimicrobial agents are able to bind the bacteria thereby avoiding the adhesion process on the mucosa! cells. However, these antimicrobial agents are not able to remove the biofilm.
The biofilm phenotype of some species has been shown to differ radically from the planktonic phenotype of the same organism. One of the facets in which biofilm bacteria differ the most profoundly from their planktonic counterparts, is in the critical matter of resistance to antibacterial agents. Results of in vitro studies showed Staphylococcus epidermis and Staphylococcus aureus were significantly more sensitive to the Lactoperoxidase system where the microorganisms are under planktonic cells than the biofilm cells, since the number of viable planktonic cells decrease by approximately 6 log units compared to a reduction of 1 log units or less in the number of biofilm cells.
The test results on the total bacteria count confirm that biofilm cells are more resistant than planktonic cells. This is believed to be due to a physical protection by the biofilm matrix or by an altered physiology of bacterial mode of growth.
In many cases, the bottom layer of biofilm will consist of anaerobic bacteria. As a result these biofilm cells may escape the inhibitory effect of the lactoperoxidase system, even though, under aerobic conditions, these cells have limited resistance to the lactoperoxidase system. These biofilm cells also escape the inhibitory effect of the lactoferrin, lysozyme and/or immunoglobulins.
Secretion liquids have long been known to be active against a number of bacteria, viruses, yeast and protozoa. But, saliva supplementation with thiocyanate/peroxidase/H2O2 systems has been shown to be ineffective in vivo on the salivary bacterial count. Formulation containing a combination of growth factor and saliva antibacterials has been described for use against biofilms adherent to cell surface. For instance, WO 01/03727 describes the use of a composition comprising a combination of a growth factor and at least one compound selected from peroxidase, lactoferrin, lactoferrin peptides, lysozyme and immunoglobulins for the prophylaxis and therapy of infectious diseases caused by microorganisms present in biofilms.
However, there still remains a need for improved compositions useful for the prophylaxis and/or therapy of diseases caused by microorganisms and in particular for the control of biofilms, thereby limiting the occurrence and progression of infectious diseases. It is the main object of the present invention to provide a composition effective for the prophylaxis and/or therapy of diseases caused by microorganisms and in particular it is another object of the present invention to provide a composition effective against planktonic microorganism and microorganism organized in biofilms, i.e. which compositions are able to disinfect surfaces.